Systematic development of a highly efficient cell factory for 5-aminolevulinic acid production

• Published in: Trends in biotechnology (Regular ed.)
• Main Authors: Zhou, Houming, Zhang, Chengyu, Li, Zilong, Xia, Menglei, Li, Zhenghong, Wang, Zhengduo, Tan, Gao-Yi, Luo, Ying, Zhang, Lixin, Wang, Weishan
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 06.08.2024
• Subjects: 5-aminolevulinic acid; artificial homeostasis; collaborative optimization; dynamic regulation; enzyme mining; systems metabolic engineering; targets combination; targets combination; dynamic regulation; systems metabolic engineering; artificial homeostasis; enzyme mining; 5-aminolevulinic acid; collaborative optimization
• ISSN: 0167-7799; 1879-3096; 1879-3096
• DOI: 10.1016/j.tibtech.2024.06.004

Establishment of a highly efficient cell factory is imperative for 5-aminolevulinic acid (5-ALA) biomanufacturing.A streamlined workflow is described that enables highly efficient 5-ALA synthase mining.Genome-scale model-guided identification and combination of multiplex targets are reported.An artificial homeostasis was designed for dynamically responding to, and fine-tuning, redox status.Final collaborative optimization resulted in the highest 5-ALA biomanufacturing performance achieved to date. The versatile applications of 5-aminolevulinic acid (5-ALA) across the fields of agriculture, livestock, and medicine necessitate a cost-efficient biomanufacturing process. In this study, we achieved the economic viability of biomanufacturing this compound through a systematic engineering framework. First, we obtained a 5-ALA synthase (ALAS) with superior performance by exploring its natural diversity with divergent evolution. Subsequently, using a genome-scale model, we identified and modified four key targets from distinct pathways in Escherichia coli, resulting in a final enhancement of 5-ALA titers up to 21.82 g/l in a 5-l bioreactor. Furthermore, recognizing that an imbalance of redox equivalents hindered further titer improvement, we developed a dynamic control system that effectively balances redox status and carbon flux. Ultimately, we collaboratively optimized the artificial redox homeostasis system at the transcription level with other cofactors at the feeding level, demonstrating the highest recorded performance to date with a titer of 63.39 g/l for the biomanufacturing of 5-ALA. The versatile applications of 5-aminolevulinic acid (5-ALA) across the fields of agriculture, livestock, and medicine necessitate a cost-efficient biomanufacturing process. In this study, we achieved the economic viability of biomanufacturing this compound through a systematic engineering framework. First, we obtained a 5-ALA synthase (ALAS) with superior performance by exploring its natural diversity with divergent evolution. Subsequently, using a genome-scale model, we identified and modified four key targets from distinct pathways in Escherichia coli, resulting in a final enhancement of 5-ALA titers up to 21.82 g/l in a 5-l bioreactor. Furthermore, recognizing that an imbalance of redox equivalents hindered further titer improvement, we developed a dynamic control system that effectively balances redox status and carbon flux. Ultimately, we collaboratively optimized the artificial redox homeostasis system at the transcription level with other cofactors at the feeding level, demonstrating the highest recorded performance to date with a titer of 63.39 g/l for the biomanufacturing of 5-ALA. Graphical abstract [Display omitted] The economic viability of biomanufacturing 5-aminolevulinic acid (5-ALA) was successfully demonstrated in this study, showcasing excellent production performance. The titer reached a record-breaking 63.39 g/l at 44 h, representing the highest reported value to date, with the productivity of 1.44 g/l/h. Although the yield (0.384 mol/mol glucose) was lower than theoretically expected, the significant value of 5-ALA positions our developed cell factory competitively for efficient industrial-scale biomanufacturing. Therefore, no challenges unique to this compound can be identified for full-scale fermentation, particularly considering that our 5-ALA cell factory was derived from a widely utilized Escherichia coli strain. Currently, two primary approaches are used to produce 5-ALA: chemical synthesis and biomanufacturing. Microbial biosynthesis of 5-ALA presents a more facile, environmentally benign, and low-cost alternative. Given that 5-ALA is a non-protein amino acid, we anticipated that the entire biomanufacturing process would be analogous to the biomanufacturing of canonical amino acids. Therefore, although a separation process was not implemented in this study, we believe that our developed 5-ALA cell factory exhibits excellent parameters and represents a highly cost-efficient biomanufacturing process. Within NASA’s Technology Readiness Level (TRL) system, we propose that this 5-ALA cell factory has reached TRL 6, indicating a fully functional prototype suitable for demonstration in real production scenarios. A systematic engineering framework was demonstrated to construct a 5-aminolevulinic acid (5-ALA) cell factory, achieving the economic viability of biomanufacturing this compound with an unprecedented production performance (63.39 g/l). This comprehensive framework encompasses enzyme mining, multitarget engineering, artificial homeostasis design, and collaborative optimization.